Severe hypofibrinogenemia in patients bitten by Gloydius tsushimaensis in Tsushima Island, Nagasaki, Japan, and treatment strategy.

Gloydius tsushimaensis is an endemic species inhabiting only Tsushima, a remote Japanese island, and is a distinct species from Gloydius blomhoffii widely distributed throughout mainland Japan and Gloydius brevicaudus and Gloydius ussuriensis which are geographically distributed in South Korea. This is the first multicenter retrospective study of G. tsushimaensis bites in Japan. A study of seventy-two patients who visited the former Izuhara Hospital, the former Naka Tsushima Hospital, Tsushima Hospital, and Kamitsushima Hospital during the fourteen years from January 1, 2005, to December 31, 2018, revealed the typical clinical characteristics of G. tsushimaensis bites. Five out of seventy-two cases (6.9%) showed severe hypofibrinogenemia, in which fibrinogen levels were below 100 mg/dl, which is an unreported clinical finding for G. blomhoffii bites. Generally, when fibrinogen levels are lower than 100 mg/dl, the bleeding risk increases, and it is perilous. Severe hypofibrinogenemia cases did not improve after G. blomhoffii antivenom administration. Additionally, all five cases had disseminated intravascular coagulation, and there were two cases of acute kidney injury and one death. five cases had a median maximum creatine kinase level of 5171 IU/l (Interquartile range: 4992-41,310). Although the mechanism is not precise, coagulation tests showed that the G. tsushimaensis venom contains a thrombin-like enzyme. Based on this research, we created an algorithm for the treatment of G. tsushimaensis bites and unified the treatment methods used on the island.

Phagostimulatory effect of uptake of PLGA microspheres loaded with rifampicin on alveolar macrophages.

Our previous results on the phagocytic activity of alveolar macrophages (Mϕs) toward poly(lactic-co-glycolic) acid microspheres (PLGA MS) loaded with the anti-tuberculosis agent rifampicin (R-PLGA MS) suggest that the phagocytosis of R-PLGA MS enhances the phagocytic activity of Mϕ cells. To confirm this possibility, we examined the effect of phagocytosis of R-PLGA MS and polystyrene latex (PSL) MS on the phagocytic uptake of fluorescent PSL (F-PSL) MS by cells of the rat alveolar macrophage cell line NR8383 at 37°C. Phagocytic activity was examined in terms of the population of Mϕ cells that had phagocytosed MS (N(total)) and the total number of MS phagocytosed (n(total)) by counting the phagocytic Mϕ cells and the MS ingested in optical microscopic fields. Phagocytosis of R-PLGA MS enhanced about 1.5 times the values of N(total) and n(total) of the phagocytosis of F-PSL MS under the conditions where the phagocytosis of F-PSL MS did not attain the saturated level. In contrast, the phagocytosis of PSL MS did not enhance the phagocytic activity of Mϕ cells toward F-PSL MS. In conclusion, R-PLGA MS are favorable for drug delivery of anti-tuberculosis agents into alveolar Mϕs due to their ability to up-regulate the phagocytosis of MS.

Delivery of rifampicin-PLGA microspheres into alveolar macrophages is promising for treatment of tuberculosis.

Inhalation delivery of poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with the anti-tuberculosis agent rifampicin (RFP-PLGA MS) to alveolar macrophage (M phi) cells could be an effective drug delivery system for the treatment of tuberculosis. To examine this possibility, we studied (1) the bactericidal effect of RFP-PLGA MS on Mycobacterium bovis Bacillus Calmette-Guérin (BCG)-infected rat alveolar M phi NR8383 cells, and (2) changes in the biochemical events induced in these cells by the uptake of RFP-PLGA MS. The amount of intracellular RFP imported into the M phi s by RFP-PLGA MS containing 0.25 and 2.50 microg RFP/mL was more than twice and ten times, respectively, than that attained with 5.00 microg/mL of RFP solution; and the MS exerted more potent bactericidal effect on BCG inside M phi cells than 5.00 microg RFP/mL solution after incubation for 7 days. RFP-PLGA MS little affected the viability of M phi cells, whereas the polystyrene latex (PSL) MS used as a reference decreased it significantly. RFP-PLGA MS did not stimulate the production of tumor necrosis factor-alpha (TNF-alpha), nitric oxide, interleukin-10 (IL-10), and transforming growth factor-beta1 (TGF-beta1) by the M phi cells, whereas PSL MS stimulated all of these mediators except IL-10. We conclude that RFP-PLGA MS are bio-safe microspheres due to their "silent" nature when taken into M phi cells and that they are promising for the treatment of tuberculosis by pulmonary inhalation.

Exact determination of phagocytic activity of alveolar macrophages toward polymer microspheres by elimination of those attached to the macrophage membrane.

A method for exact determination of phagocytic activity of alveolar macrophage (Mvarphi) cells toward synthetic microspheres (MS) by optical microscopy was developed. We examined the effectiveness of the treatment of Mvarphi samples with trypsin, acid or xylene to remove the polystyrene latex microspheres (PSL MS) attached to Mvarphi cell membranes during their phagocytosis by Mvarphi cells. We found that centrifugation, which was employed to collect Mvarphi samples after incubation with MS, affected significantly the efficiency of the various treatments. Of the three treatments, xylene treatment without centrifugation was the most effective to determine the phagocytic activity of Mvarphi cells, as xylene dissolved the PSL MS on the cell surface almost completely. This treatment was also effective in the case of poly(lactic-co-glycolic acid) MS (PLGA MS), which have been commonly used as an efficient vehicle for drug delivery system.

Phagocytic activity of alveolar macrophages toward polystyrene latex microspheres and PLGA microspheres loaded with anti-tuberculosis agent.

Phagocytosis of alveolar macrophages (Mphis) toward poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) loaded with the anti-tuberculosis agent rifampicin (RFP-PLGA MS) has been shown to be effective for the treatment of tuberculosis. The phagocytosis should be evaluated in terms of that toward reference MS. We chose polystyrene latex (PSL) MS as a reference. In this study, phagocytic activity of cell line NR8383, derived from rat alveolar Mphi, toward PSL MS with various diameters was examined by incubating the cells for 4h at 37 degrees C with various numbers of PSL MS per Mphi cell (MS/Mphi=0.1-10). The results were then compared with those of the phagocytosis toward RFP-PLGA MS. We determined the phagocytic activity by counting the population of Mphi cells that had phagocytosed MS (N) and the number of particles phagocytosed (n) in microscopic fields. Both N and n for PSL and RFP-PLGA MS increased in general with an increase in MS/Mphi, but both of these values for PSL MS were smaller than those for RFP-PLGA MS. Phagocytosis of the particles were dependent on the particle size; i.e., of the PSL MS the 6-mum ones were taken up by Mphi the most, and the RFP-PLGA MS 3 microm in diameter seemed to be phagocytosed the most efficiently, although we were not able to determine exactly the phagocytosis of 6- and 10-microm RFP-PLGA MS. From the changes in N and n values with MS/Mphi, the phagocytosis of RFP-PLGA MS was likely to enhance the phagocytic activity of Mphi cells, but this effect did not seem to be significant for PSL MS.

Optimum conditions for efficient phagocytosis of rifampicin-loaded PLGA microspheres by alveolar macrophages.

We examined the phagocytic activities of alveolar macrophages (NR8383 cells) toward poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with the anti-tuberculosis agent rifampicin (RFP), the sizes of which were between 1 microm and 10 microm. We found that 1) the phagocytosis was dependent greatly on the particle size and the number of particles added; 2) macrophages phagocytosed considerably the PLGA microspheres loaded with RFP, the diameter of which was between 1 microm and 6 microm, but took up few 10-microm particles; 3) the population of the macrophages that phagocytosed 1-microm or 3-microm particles was larger than that of those phagocytosed 6- or 10-microm particles; 4) a considerable population of macrophages were not able to phagocytose even the 1- and 3-microm particles; 5) the most efficient deliveries of RFP into each macrophage cell and a large population of macrophages were achieved by the phagocytosis of 3-microm particles; and 6) phagocytosis did not affect macrophage viability in 4 h after the start of phagocytosis.